Inhibition of hendra virus fusion.

Hendra virus (HeV) is a recently identified paramyxovirus that is fatal in humans and could be used as an agent of bioterrorism. The HeV receptor-binding protein (G) is required in order for the fusion protein (F) to mediate fusion, and analysis of the triggering/activation of HeV F by G should lead to strategies for interfering with this key step in viral entry. HeV F, once triggered by the receptor-bound G, by analogy with other paramyxovirus F proteins, undergoes multistep conformational changes leading to a six-helix bundle (6HB) structure that accomplishes fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions (HRN and HRC) near the fusion peptide and the transmembrane domains, respectively. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing F, after the initial triggering step, from forming the 6HB structure that is required for fusion. HeV peptides have previously been found to be effective at inhibiting HeV fusion. However, we found that a human parainfluenza virus 3 F-peptide is more effective at inhibiting HeV fusion than the comparable HeV-derived peptide.

Human parainfluenza virus type 3 HN-receptor interaction: effect of 4-guanidino-Neu5Ac2en on a neuraminidase-deficient variant.

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity. We have now examined this mechanism, as well as neuraminidase's role in the viral life cycle, using a neuraminidase-deficient HPF3 variant (C28a) and stable cell lines expressing C28a or wild-type (wt) HN. C28a, which has a wt F sequence and two point mutations in the HN gene corresponding to two amino acid changes in the HN protein, is the first HPF3 variant with insignificant neuraminidase activity. Cells expressing C28a HN did not bind erythrocytes at 4 degrees C unless pretreated with neuraminidase, but no such pretreatment was required for hemadsorption activity (HAD) at 22 or 37 degrees C. HAD was blocked by 4-GU-DANA, attesting to the ability of this compound to inhibit HN's receptor-binding activity. C28a or wt plaque enlargement, a process that involves cell-cell fusion and does not depend on virion release, is diminished by the presence of 4-GU-DANA, confirming the inhibitory effect of 4-GU-DANA on the fusogenic function of C28a HN. In C28a-infected cell monolayers, virion release and thus multicycle replication are severely restricted. This defect was corrected by supplementation of exogenous neuraminidase and also by the addition of 4-GU-DANA; neuraminidase destroys the receptors whereby newly formed C28a virions would remain attached to the cell surface, whereas 4-GU-DANA prevents the attachment itself, obviating the need for receptor cleavage. In accord with the ability of 4-GU-DANA to prevent attachment, the neuraminidase inhibitory effect of 4-GU-DANA on wt HPF3 did not diminish virion release into the medium. Thus, it is by inhibition of viral entry and syncytium formation that sialic acid analogs like 4-GU-DANA may counteract wt HPF3 infection.

A single amino acid alteration in the human parainfluenza virus type 3 hemagglutinin-neuraminidase glycoprotein confers resistance to the inhibitory effects of zanamivir on receptor binding and neuraminidase activity.

Entry and fusion of human parainfluenza virus type 3 (HPF3) requires interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (4-GU-DANA; zanamivir), a sialic acid transition-state analog designed to fit the influenza virus neuraminidase catalytic site, possesses antiviral activity at nanomolar concentrations in vitro. We have shown previously that 4-GU-DANA also inhibits both HN-mediated binding of HPF3 to host cell receptors and HN's neuraminidase activity. In the present study, a 4-GU-DANA-resistant HPF3 virus variant (ZM1) was generated by serial passage in the presence of 4-GU-DANA. ZM1 exhibited a markedly fusogenic plaque morphology and harbored two HN gene mutations resulting in two amino acid alterations, T193I and I567V. Another HPF3 variant studied in parallel, C-0, shared an alteration at T193 and exhibited similar plaque morphology but was not resistant to 4-GU-DANA. Neuraminidase assays revealed a 15-fold reduction in 4-GU-DANA sensitivity for ZM1 relative to the wild type (WT) and C-0. The ability of ZM1 to bind sialic acid receptors was inhibited 10-fold less than for both WT and C-0 in the presence of 1 mM 4-GU-DANA. ZM1 also retained infectivity at 15-fold-higher concentrations of 4-GU-DANA than WT and C-0. A single amino acid alteration at HN residue 567 confers these 4-GU-DANA-resistant properties. An understanding of ZM1 and other escape variants provides insight into the effects of this small molecule on HN function as well as the role of the HN glycoprotein in HPF3 pathogenesis.

The anti-influenza virus agent 4-GU-DANA (zanamivir) inhibits cell fusion mediated by human parainfluenza virus and influenza virus HA.

4-GU-DANA (zanamivir) (as well as DANA and 4-AM-DANA) was found to inhibit the neuraminidase activity of human parainfluenza virus type 3 (HPF3). The viral neuraminidase activity is attributable to hemagglutinin-neuraminidase (HN), an envelope protein essential for viral attachment and for fusion mediated by the other envelope protein, F. While there is no evidence that HN's neuraminidase activity is essential for receptor binding and syncytium formation, we found that 4-GU-DANA prevented hemadsorption and fusion of persistently infected cells with uninfected cells. In plaque assays, 4-GU-DANA reduced the number (but not the area) of plaques if present only during the adsorption period and reduced plaque area (but not number) if added only after the 90-min adsorption period. 4-GU-DANA also reduced the area of plaques formed by a neuraminidase-deficient variant, confirming that its interference with cell-cell fusion is unrelated to inhibition of neuraminidase activity. The order-of-magnitude lower 50% inhibitory concentrations of 4-GU-DANA (and also DANA and 4-AM-DANA) for plaque area reduction and for inhibition in the fusion assay than for reducing plaque number or blocking hemadsorption indicate the particular efficacy of these sialic acid analogs in interfering with cell-cell fusion. In cell lines expressing influenza virus hemagglutinin (HA) as the only viral protein, we found that 4-GU-DANA had no effect on hemadsorption but did inhibit HA2b-red blood cell fusion, as judged by both lipid mixing and content mixing. Thus, 4-GU-DANA can interfere with both influenza virus- and HPF3-mediated fusion. The results indicate that (i) in HPF3, 4-GU-DANA and its analogs have an affinity not only for the neuraminidase active site of HN but also for sites important for receptor binding and cell fusion and (ii) sialic acid-based inhibitors of influenza virus neuraminidase can also exert a direct, negative effect on the fusogenic function of the other envelope protein, HA.

Mechanism of interference mediated by human parainfluenza virus type 3 infection.

Viral interference is characterized by the resistance of infected cells to infection by a challenge virus. Mechanisms of viral interference have not been characterized for human parainfluenza virus type 3 (HPF3), and the possible role of the neuraminidase (receptor-destroying) enzyme of the hemagglutinin-neuraminidase (HN) glycoprotein has not been assessed. To determine whether continual HN expression results in depletion of the viral receptors and thus prevents entry and cell fusion, we tested whether cells expressing wild-type HPF3 HN are resistant to viral infection. Stable expression of wild-type HN-green fluorescent protein (GFP) on cell membranes in different amounts allowed us to establish a correlation between the level of HN expression, the level of neuraminidase activity, and the level of protection from HPF3 infection. Cells with the highest levels of HN expression and neuraminidase activity on the cell surface were most resistant to infection by HPF3. To determine whether this resistance is attributable to the viral neuraminidase, we used a cloned variant HPF3 HN that has two amino acid alterations in HN leading to the loss of detectable neuraminidase activity. Cells expressing the neuraminidase-deficient variant HN-GFP were not protected from infection, despite expressing HN on their surface at levels even higher than the wild-type cell clones. Our results demonstrate that the HPF3 HN-mediated interference effect can be attributed to the presence of an active neuraminidase enzyme activity and provide the first definitive evidence that the mechanism for attachment interference by a paramyxovirus is attributable to the viral neuraminidase.

The use of a quantitative fusion assay to evaluate HN-receptor interaction for human parainfluenza virus type 3.

Sialic acid is the receptor determinant for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, HN must interact with its receptor. In addition to its role in receptor binding and fusion promotion, the HPF3 HN molecule contains receptor-destroying (sialidase) activity. The putative active sites are in the extracellular domain of this type II integral membrane protein. However, HN is not available in crystalline form; the exact locations of these sites, and the structural requirements for binding to the cellular receptor, which has not yet been isolated, are unknown. Nor have small molecular synthetic inhibitors of attachment or fusion that would provide insight into these processes been identified. The strategy in the present study was to develop an assay system that would provide a measure of a specific step in the viral cycle-functional interaction between viral glycoproteins and the cell during attachment and fusion-and serve to screen a variety of substances for inhibitory potential. The assay is based on our previous finding that CV-1 cells persistently infected (p.i.) with HPF3 do not fuse with one another but that the addition of uninfected CV-1 cells, supplying the critical sialic acid containing receptor molecules that bind HN, results in rapid fusion. In the present assay two HeLa cell types were used: we persistently infected HeLa-LTR-betagal cells, assessed their fusion with uninfected HeLa-tat cells, and then quantitated the beta-galactosidase (betagal) produced as a result of this fusion. The analog alpha-2-S-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2SMe) interfered with fusion, decreasing betagal production by 84% at 50 mM and by 24% at 25 mM. In beginning to extend our studies to different types of molecules, we tested an unsaturated derivative of sialic acid, 2,3-dehydro-2-deoxy-n-acetyl neuraminic acid (DANA), which is known to inhibit influenza neuraminidase by virtue of being a transition-state analog. We found that 10 mM DANA inhibited neuraminidase activity in HPF3 viral preparations. More significantly, this compound was active in our assay of HN-receptor interaction; 10 mM DANA completely blocked fusion and betagal production, and hemadsorption inhibition by DANA suggested that DANA blocks attachment. In plaque reduction assays performed with the compounds, the active analog alpha-Neu5thioAc2SMe reduced plaque formation by 50% at a 50 mM concentration; DANA caused a 90% inhibition in the plaque reduction assay at a concentration of 25 mM. Our results indicate that specific sialic acid analogs that mimic the cellular receptor determinant of HPF3 can block virus cell interaction and that an unsaturated n-acetyl-neuraminic acid derivative with affinity to the HN site responsible for neuraminidase activity also interferes with HN-receptor binding. Strategies suggested by these findings are now being pursued to obtain information regarding the relative locations of the active sites of HN and to further elucidate the relationship between the receptor-binding and receptor-destroying activities of HN during the viral life cycle. The quantitative assay that we describe is of immediate applicability to large-scale screening for potential inhibitors of HPF3 infection in vivo.

Enzymic composition and growth rate of human pleural mesothelioma transplants in nude mice.

The enzymic composition of 7 human mesothelioma lines propagated in nude mice was compared with 4 of the original and 15 additional mesotheliomas sampled during the patients' surgery. The xenografts exhibited several-fold higher thymidine kinase (TK), uridine kinase (UK), phosphoserine phosphatase (PSP) and peptidyl proline hydroxylase (PPH) concentrations than the fresh human samples, while their DNA, gamma-glutamyl transpeptidase (GGT) and beta-galactosidase (Bgal) contents remained similar. The volume growth rate of the xenografts (doubling time, DT = 9.23 +/- 1.25 days) was much faster than that of tumors in the human host, and the decline of this rate with increasing nodule size was accompanied by decreases in TK and PSP concentrations. This first quantitative biochemical study of xenografted human neoplasms indicates that 1) pleural mesotheliomas, though preserving their histological characteristics after heterotransplantation, show considerable increases of enzymes in nucleic acid, collagen, and nonessential amino acid synthesis, and that 2) the concentration of TK is a good indicator of the different growth properties of tumors in a mouse rather than in the human host.

Neutrophil maturation and hypersegmentation promoted in normal bone marrow by a carcinoma-elaborated protein factor.

In rats with subcutaneously transplanted mammary carcinoma 5A (MC) and 2 to 10-fold elevations in the blood content of mature neutrophils, 30-50% of the neutrophils showed pronounced hypersegmentation. This phenomenon could be reproduced in liquid culture of bone marrow cells from normal (tumor-free) animals by 48 hr incubation with the MC host's serum, or with MC-conditioned medium whose activity was attributable to an over 50,000 MW protein. The effects in vitro, occurring without change in total cell number and accompanied by increases in gamma-glutamyltranspeptidase (GGT) and alkaline phosphatase (AP) activity, included decreases in the percents of progenitors (myeloblasts, promyelocytes, myelocytes, metamyelocytes and bands) and an increase in mature neutrophils 50% of which exhibited obvious hypersegmentation. Much less if any neutrophil hypersegmentation, and no statistically significant decrease in immature cells, occurred in response to the several colony stimulating factors (CSFs) tested, although (in addition to inducing GGT and AP) some CSFs did cause an increase in mature neutrophils. These investigations demonstrate the efficacy of a MC-elaborated blood-borne protein to promote myeloid cell maturation, and describe a system for the first time in which neutrophil hypersegmentation can be studied in vitro.

gamma-Glutamyltranspeptidase activity in intact leukocytes: flow cytometric analysis and sorting.

A fluorescent method developed for visualizing gamma-glutamyltranspeptidase (GGT) in intact liver cells was adapted to leukocytes and used in a multiparameter flow cytometric study of blood and bone marrow cells from rats with subcutaneous implants of mammary carcinoma 5A. The severe granulocytosis caused by this non-metastatic tumor was preceded by a progressive rise in the percentage of leukocytes with high GGT fluorescence. Both granulocytes and small, immature cells of bone marrow showed increased GGT expression, whereas in blood this increase was attributable entirely to mature granulocytes. At 28 days (but not yet at 14 days) after carcinoma implantation, 20-30% of blood or bone marrow granulocytes constituted a distinct subpopulation in that their GGT fluorescence intensity range was much higher and did not overlap with the range for the rest of the population. The results indicate that fluorescent GGT assay of intact leukocytes provides a useful probe for flow cytometric analysis of population heterogeneity in leukoproliferative disorders.

Mitogen activation of human lymphocytes from normal and acute lymphocytic leukemia (ALL) subjects.

Lymphocytes from acute lymphocytic leukemia (ALL) subjects were converted by mitogens to blast-like cells whose microscopic appearance and rate of formation was indistinguishable from those in mitogen incubated control lymphocytes. In ALL lymphocytes, however, pokeweed mitogen (PWM) failed to stimulate GGT expression; the mean increase it caused in thymidine kinase (TK) activity and thymidine incorporation was normal, though there were appreciable individual variations. These variations were also apparent with concanavalin A (Con A) but, in most ALL cases, TK and thymidine incorporation rose to much higher levels than in Con-A-treated control lymphocytes. The results indicate that evaluation of the response to mitogens by quantitative biochemical criteria provides a sensitive method for revealing functional impairments in microscopically normal ALL lymphocytes.

Phenylalanine uptake in neonatal and infant rat brain.

At the same severe elevations in blood phenylalanine (Phe) levels maintained for 4 h, much higher cerebral Phe concentrations were found in 4-day-old than in 16- or 70-day-old rats. In order to compare this developmental change with 14C-Phe influx mediated by the L transport system, the rapid intracarotid injection method was adapted for use in neonatal rats. The brain uptake index (BUI) thus determined for the first time through the suckling period was significantly higher on the 4th day of age than on the 7th or 24th day, while no significant change occurred during subsequent life. This early period of change in influx across the blood-brain barrier overlapped with the age period of decrease of the hyperphenylalaninemia-associated accumulation of Phe in the brain. The results indicate that by the time when intermittent feeding begins, the brain has developed a considerable ability (a) to protect itself against physiological (e.g. postprandial) fluctuations in circulating Phe levels, and (b) to restrict the cerebral accumulation of Phe from pathologically elevated blood concentrations such as those in phenylketonuria.

Enhancement of gamma-glutamyl transpeptidase expression in bone marrow cells by colony stimulating factors.

Studies on blood serum from mammary carcinoma (MC) hosts, which promoted gamma-glutamyl transpeptidase (GGT) expression by normal rat bone marrow cells in liquid culture, were extended to various granulocyte-macrophage colony stimulating factors (CSFs). GGT concentration per cell was found to increase (without change in total cell number) by incubation for 48 h with purified CSF-2 gamma and CSF-1 (but not interleukin-3), with human giant cell elaborated GM-CSF and L-cell conditioned medium, as well as with the 3 MC preparations (host serum, MC extract and MC conditioned medium). GGT-inducing ability (per milligram protein) ranked the 7 preparations in the same order as did their proliferative effect (number of colonies per milligram protein) in the standard mouse bone marrow agar culture system. The quantitative correlation between these two kinds of activities (linear for their logarithmic values) was highly significant, r = 0.976, p less than 0.001. The alkaline phosphatase concentration of bone marrow cells in liquid culture was also increased in the presence of the same 7 preparations, and this again was proportional (r = 0.985, p less than 0.001) to their colony stimulating potential.

Characterization of a mammary carcinoma elaborated factor stimulating gamma-glutamyltranspeptidase expression in bone marrow cells.

The elevation of bone marrow gamma-glutamyltranspeptidase (GGT) and alkaline phosphatase (AP) content in rats carrying mammary carcinoma 5A (MC), reproduced in a short-term (48-h) liquid culture of normal bone marrow cells, was found to be attributable to a blood-borne protein factor with an apparent molecular weight of 60,000. Partial purification, based on the extent of stimulation of GGT expression in this culture, increased the specific activity of the host serum from 1.5 to 40 units and that of MC extracts from 6 to 560 units. Production of the factor by MC in vitro, however, resulted in specific activities of 3000 units in the conditioned medium, and a further 60-fold purification was achieved by DEAE-cellulose, Sephadex G-100, and hydroxylapatite chromatography. The chemical characteristics of the MC-elaborated protein indicate nonidentity to previously isolated colony formation stimulating factors which also induced GGT (and AP) expression by rat bone marrow cells. Most of the AP inducing ability of the MC-serum and MC-conditioned medium copurified with and was still present in preparations with the highest specific activity vis à vis GGT. In mouse (instead of rat) bone marrow cells, however, no AP response accompanied the stimulation of GGT expression by MC (or colony formation stimulating factor) preparations.

Diminished lymphocyte and granulocyte gamma-glutamyltranspeptidase activity in acute lymphocytic leukemia and response to chemotherapy.

gamma-Glutamyltranspeptidase (GGT) activity (per mg protein) in blood lymphoid cells of 27 children with acute lymphoblastic leukemia (ALL) (1.05 +/- 0.15) was significantly below that of controls (2.25 +/- 0.30), became normalized during chemotherapy-induced remission (2.47 +/- 0.26), and was low again (1.59 +/- 0.62) in relapsed subjects. Individual variations in the GGT activity of the blood lymphoid cell fraction (per mg protein) bore a significant inverse correlation to the number of white blood cells (WBC) as well as of blasts per ml blood. Blasts had minimal GGT activity; however, partial GGT deficiency was also exhibited by the microscopically normal circulatory lymphocytes of several patients prior to treatment and in relapsed subjects whose blood was still devoid of blasts. Significantly diminished GGT activity (per mg protein) was found in the blood granulocytes of ALL subjects. This deficit, restored during remission and present again at relapse, varied in magnitude but showed no statistically significant correlation to the different patients' degree of neutropenia. In about one-third of the newly diagnosed or relapsed pre-B ALL children, the circulatory granulocytes' GGT activity was only 10-20% of normal. The results suggest that 1) the presence or absence of this sign of functional maldifferentiation in granulocytes is a factor in the heterogeneity of disease manifestation among subjects with apparently the same type of ALL and that 2) measurement of GGT in the circulatory granulocytes, as well lymphocytes, may be useful for monitoring the efficacy of chemotherapy.

Enzyme pathology of human mesotheliomas.

In samples of 16 surgically resected mesotheliomas arising from the pleura of the human lung, 6 enzymes from different metabolic pathways, DNA, and mitotic frequency were quantified. The mesotheliomas, irrespective of cell type or grade, showed lower gamma-glutamyl transpeptidase (GGT) concentration than 36 of the 38 pulmonary adenocarcinomas. The mean concentration of this enzyme in the 15 mesotheliomas was an eighth of that in the 56 carcinomas, whereas their DNA content was similar. The quantitative correlation of thymidine kinase (TK), uridine kinase (UK), and phosphoserine phosphatase to mitotic frequency was highly significant for mesotheliomas, as well as for carcinomas. As estimated from their TK [and its recently established quantitative correlation to volume doubling time (DT)], the DT of the 16 mesotheliomas ranged from 50 to over 700 days, with a somewhat longer median than the median for pulmonary carcinomas. Subject survival, though shortest for the 2 sarcomatous mesothelioma cases, varied over an overlapping range for mesotheliomas with epithelial or mixed cell type. The biopsy samples' TK and UK concentrations, however, showed a significant inverse correlation with months of survival after diagnosis. Survival time after the first appearance of symptoms decreased linearly (on log scales) with TK concentration (P less than .001) over the 14 cases. The results of this first quantitative study of a spectrum of biochemical constituents of mesotheliomas identify GGT as an enzyme whose measurement guards against mistaking mesotheliomas and adenocarcinomas for one another and show that the TK concentrations of these mesothelioma samples bear a highly significant, inverse correlation to the postdiagnosis survival time of the individual subjects.

Cerebral serotonin regulation by phenylalanine analogues and during hyperphenylalaninemia.

Severe hyperphenylalaninemia induced in infant rats by 3 days of treatment with p-chlorophenylalanine (p-cl phe) plus phenylalanine (phe) did not lower the tryptophan concentration of the brain, and the cerebral serotonin (5-HT) deficiency was attributable entirely to the known suppression to tryptophan hydroxylase (TPH) by p-cl phe. The decrease in 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) was thus no more pronounced than in rats which, treated with p-cl phe alone, were devoid of hyperphenylalaninemia. Suppression of TPH was found to also underlie the decrease in cerebral 5-HT caused by treatment with alpha-methylphenylalanine (alpha-mephe) alone: a 22% loss of midbrain TPH activity was detectable 24 hr after an injection only, reverted toward the normal during the next 2 days, and was clearly unrelated to the weak competitive inhibition of the enzyme by alpha-mephe in vitro. However, alpha-mephe (unlike p-cl phe), when administered together with phe, did not suppress TPH, nor did it counterbalance the reduction of cerebral tryptophan uptake by excess phe. Thus the 5-HT diminution in the rat model of phenylketonuria produced by treatment with alpha-mephe plus phe was attributable to hyperphenylalaninemia and the inhibition of tryptophan transport to the brain. Injection of tryptophan was found to restore the cerebral 5-HT level in the face of persistently severe hyperphenylalaninemia.

Cerebral glycine content and phosphoserine phosphatase activity in hyperaminoacidemias.

Chronic hyperphenylalaninemia maintained with the aid of a suppressor of phenylalanine hydroxylase, alpha-methylphenylalanine, increases the glycine concentration and the phosphoserine phosphatase activity of the developing rat brain but not that of liver or kidney. Similar increases occur after daily injections with large doses of phenylalanine alone, while tyrosine, isoleucine, alanine, proline, and threonine, were without effect. Treatment with methionine, which increases the phosphoserine phosphatase activity of the brain and lowered that of liver and kidney, left the cerebral glycine level unchanged. When varying the degrees of gestational or early postnatal hyperphenylalaninemia, a significant linear correlation was found between the developing brains' phosphoserine phosphatase and glycine concentration. Observations on the uptake of injected glycine and its decline further indicate that coordinated rises in the brain's phosphoserine phosphatase and glycine content associated with experimental hyperphenylalaninemia denote a direct impact of phenylalanine on the intracellular pathway of glycine synthesis in immature animals.

Lymphocyte thymidine kinase and treatment response in acute lymphocytic leukemia.

The activity of thymidine kinase (TK) and the proportion of its isozymes (TK1/TK2) were studied in peripheral lymphoid cells of 37 children with acute lymphocytic leukemia (ALL). The high TK in 25 untreated subjects (31.5 +/- 8.9) decreased during chemotherapy-induced remission to uniformly low (5.3 +/- 0.4) normal values, and rose again during relapse to a mean of (24.8 +/- 8.1). The proportion of isozyme 1 followed the same pattern but TK was a more sensitive indicator of disease state. The lymphocyte fractions' TK (per mg protein) correlated with the number (per ml blood) of WBCs, blasts and lymphocytes. Although the higher TK of blasts than of apparently normal lymphocytes was confirmed in cases permitting clean physical separation, the lymphocyte fraction of several untreated subjects with minimal blast counts also exhibited elevated TK. Moreover, this elevation was also seen in relapsed cases even if their blood (unlike bone marrow) was devoid of blasts. The results indicate that quantification of TK can reveal a subpopulation of maldifferentiated lymphocytes which are microscopically normal and that it may provide an objective parameter of prognostic differences between ALL subjects with similar hematological characteristics.

Mechanical responses of developing Fisher rat heart. Effects of steroid hormone.

Age-dependent changes in the mechanical responses of developing Fisher rat heart during the first three postnatal weeks were studied in relation to the hypothesis that the abnormality observed in the mechanical responses of the rat heart might be calcium related. Therefore the effect of frequency of stimulation as well as the response to calcium, epinephrine and ouabain on hearts of untreated and cortisol-treated rats was compared. The positive force-frequency response observed in fetal rat heart reverted to a highly negative response by the 12th to 14th postnatal day. The biphasic mechanical responses directly paralleled reported changes in circulating glucocorticoid levels in developing rat. The force-frequency response was maximally negative when the circulating levels of glucocorticoids were lowest. The reversion of the negative force-frequency responses coincided with a gradual increase reported in the circulating levels of glucocorticoids. The negative force-frequency response was absent in the cortisol-treated developing rat heart and a definite positive pattern was observed as the rats developed. A high sensitivity to free calcium concentration, seen in control fetal and and newborn hearts, diminished after the second postnatal week. By the third postnatal week, the sensitivity to high extracellular calcium concentrations was significantly reduced. The sensitivity to calcium persisted in the cortisol-treated hearts during the 3 postnatal weeks. Cortisol-treated hearts were more responsive to epinephrine than controls. The abbreviation of time to peak tension, a hallmark of the catecholamine effect, was observed at a younger age in the cortisol-treated hearts. Cortisol-treated hearts were more responsive to the inotropic effects of ouabain than controls. The possible involvement of glucocorticoids in the control of calcium handling elements of the myocardium is discussed.